Full citations for references appear at the end of the Examples section.
Peroxidases are enzymes catalyzing oxidative reactions that use H2O2 as an electron acceptor. These enzymes are widespread and occur ubiquitously in plants as isozymes that may be distinguished by their isoelectric points. Plant peroxidases contribute to the structural integrity of cell walls by functioning in lignin biosynthesis and suberization, and by forming covalent cross-linkages between extension, cellulose, pectin and other cell wall constituents (Campa, 1991). Peroxidases are also associated with plant defence responses and resistance to pathogens (Bowles, 1990; Moerschbacher 1992). Soybeans contain 3 anionic isozymes of peroxidase with a minimum Mr of 37 kDa (Sessa and Anderson, 1981). Recently one peroxidase isozyme, localised within the seed coat of soybean, has been characterized with a Mr of 37 kDa (Gillikin and Graham, 1991).
In an analysis of soybean seeds, Buttery and Buzzell (1968) showed that the amount of peroxidase activity present in seed coats may vary substantially among different cultivars. The presence of a single dominant gene Ep causes a high seed coat peroxidase phenotype (Buzzell and Buttery, 1969). Homozygous recessive epep plants are ˜100-fold lower in seed coat peroxidase activity. This results from a reduction in the amount of peroxidase enzyme present, primarily in the hourglass cells of the subepidermis (Gijzen et al., 1993). In plants carrying the Ep gene, peroxidase is heavily concentrated in the hourglass cells (osteosclereids). These cells form a highly differentiated cell layer with thick, elongated secondary walls and large intercellular spaces (Baker et al., 1987). Hourglass cells develop between the epidermal macrosclereids and the underlying articulated parenchyma, and are a prominent feature of seed coat anatomy at full maturity. The cytoplasm exudes from the hourglass cells upon imbibition with water and a distinct peroxidase isozyme constitutes five to 10% of the total soluble protein in EpEp seed coats. It is not known why the hourglass cells accumulate large amounts of peroxidase, but the sheer abundance and relative purity of the enzyme in soybean seed coats is significant because peroxidases are versatile enzymes with many commercial and industrial applications. Studies of soybean seed coat peroxidase have shown this enzyme to have useful catalytic properties and a high degree of thermal stability even at extremes of pH (McEldoon et al., 1995). These properties result in the preferred use of soybean peroxidase, over that of horseradish peroxidase, in diagnostic assays as an enzyme label for antigens, antibodies, oligonucleotide probes, and within staining techniques. Johnson et al report on the use of soybean peroxidase for the deinking of printed waste paper (U.S. Pat. No. 5,270,770; Dec. 6, 1994) and for the biocatalytic oxidation of primary alcohols (U.S. Pat. No. 5,391,488; Feb. 13, 1996). Soybean peroxidase has also been used as a replacement for chlorine in the pulp and paper industry, or as formaldehyde replacement (Freiberg, 1995).
An anionic soybean peroxidase from seed coats has been purified (Gillikin and Graham, 1991). This protein has a pI of 4.1 and Mr of 37 kDa. A method for the bulk extraction of peroxidase from seed hulls of soybean using a freeze thaw technique has also been reported (U.S. Pat. No. 5,491,085, Feb. 13, 1996, Pokara and Johnson).
Lagrimini et al (1987) disclose the cloning of a ubiquitous anionic peroxidase in tobacco encoding a protein of Mr of 36 kDa. This peroxidase has also been over expressed in transgenic tobacco plants (Lagrimini et al 1990) and Maliyakal discloses the expression of this gene in cotton (WO 95/08914).
Huangpu et al (1995) reported the partial cloning of a soybean anionic seed coat peroxidase. The 1031 bp sequence contained an open reading frame of 849 bp encoding a 283 amino acid protein with a Mr of 30,577. The Mr of this peroxidase is 7 kDa less than what one would expect for a soybean seed coat peroxidase as reported by Gillikin and Graham (1991) and possibly represents another peroxidase isozyme within the seed coat.
The upstream promoter sequences for two poplar peroxidases have been described by Osakabe et al (1995). A number of characteristic regulatory sites were identified from comparison of these sequences to existing promoter elements. Additionally, a cryptic promoter with apparent specificity for seed coat tissues was isolated from tobacco by a promoter trapping strategy (Fobert et al. 1994). The upstream regulatory sequences associated with the Ep gene in soybean are distinct from these and other previously characterized promoters. The soybean Ep promoter drives high-level expression in a cell and tissue specific manner. The peroxidase protein encoded by the Ep gene accumulates in the seed coat tissues, especially in the hour glass cells of the subepidermis. Minimal expression of the gene is detected in root tissues.
One problem arising from the desired use of soybean seed coat peroxidase is that there is variability between soybean varieties regarding peroxidase production (Buttery and Buzzell, 1986; Freiberg, 1995). Due to the commercial interest in the use of soybean seed coat peroxidase new methods of producing this enzyme are required. Therefore, the gene responsible for the expression of the 37 kDa isozyme in soybean seed coat was isolated and characterized.
Furthermore, novel regulatory regions obtained from the genomic DNA of soybean seed coat peroxidase have been isolated and characterized and are useful in directing the expression of genes of interest in seed coat tissues.